Aquatic Botany 121 (2015) 46–51 Contents lists available at ScienceDirect Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot Chloroplast dimorphism in leaves of Cabomba caroliniana (Cabombaceae) Beatriz G. Galati a,∗, Marina M. Gotelli a,b, Sonia Rosenfeldt c, Elsa C. Lattar d, G. Mónica Tourn e a Cátedra de Botánica General, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417DSE Buenos Aires, Argentina b CONICET, Argentina c Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina d Instituto de Botánica del Nordeste (UNNE-CONICET), C.C. 209, W3400CBL Corrientes, Argentina e Sede Punilla, Facultad de Agronomía, Universidad de Buenos Aires, Huerta Grande, Córdoba, Argentina article info abstract Article history: Cabomba Aublet is useful as a genetic model of angiosperm evolution. Previous anatomical studies and Received 6 February 2014 preliminary light microscopic observations on C. caroliniana A Gray revealed the presence of dimorphic Received in revised form chloroplasts in both types of leaves, floating and submerged leaves. Leaf anatomy and chloroplasts ultra- 11 November 2014 structure were analyzed and compared. The chloroplast ultrastructure of the mesophyll and epidermal Accepted 14 November 2014 cells is different. Mesophyll chloroplasts have several plastoglobuli, large starch grains and grana formed Available online 22 November 2014 by an average of 29 thylakoids. Epidermal chloroplasts are smaller, have grana formed by an average of 9 thylakoids and starch grains are occasional and smaller. A statistical analysis (t-test) was made to Keywords: Cabomba determine if differences between chloroplasts are significant. The ultrastructure of the epidermis chloro- Leaf anatomy plast is similar to the one of sun leaves chloroplasts and the ultrastructure of the mesophyll chloroplast Chloroplast ultrastructure is similar to the one of shade chloroplast observed in other species. ANA grade © 2014 Elsevier B.V. All rights reserved. 1. Introduction and B). Previous anatomical studies on Cabomba (Galati, 1981) and preliminary light microscopic observations revealed dimor- Molecular phylogenetic studies indicate that the orders phic chloroplasts in both types of leaves of C. caroliniana. The aim Amborellales, Nymphaeales and Austrobaileyales (ANA grade) of this research is to describe the leaf anatomy and to compare the diverged as separate lineages from a remaining angiosperm clade ultrastructure of the different chloroplasts present in both types of at a very early stage in flowering plant evolution. Cabomba Aublet leaves. (Nymphaeales) shows features that make it potentially useful as a genetic model of angiosperm evolution (Vialette-Guiraud et al., 2. Materials and methods 2011). It is a New World genus of aquatic plants with most species restricted to tropical or subtropical regions (Orgaard, 1991). Leaves of three different populations of C. caroliniana were Aquatic angiosperms generally have anatomical features that collected: one population was cultivated in an outdoor pond of differ from terrestrial angiosperms (Sculthorpe, 1985). A marked the Botanical Garden of the Facultad de Agronomía, Universi- foliar dimorphism is present in Cabomba (Moseley et al., 1984). dad de Buenos Aires, Buenos Aires, Argentina; another population Heterophylly is considered a result of heteroblastic development was from one natural population of INTA Delta (Latitude 59◦20 (Briggs and Walters, 1984; Kerstetter and Poeting, 1998)orof S. Longitude 34◦40 O) and the third population was from an plastic development (Titus and Sullivan, 2001). C. caroliniana A. indoor fishbowl culture. The reference material is deposited in the Gray presents floating peltate leaves with entire lamina and sub- Herbarium Gaspar Xuarez (BAA). Material was pre-fixed in 2.5% merged leaves with trichotomously dissected lamina (Galati, 1981). glutaraldehyde in phosphate buffer (pH 7.2) for 2 h and post-fixed The floating leaves are produced by flowering shoots (Fig. 1A ◦ in 1.5% OsO4 at 2 C in the same buffer for 3 h. Leaves were dehy- drated in ascending acetone series and embedded in Spurr’s resin. Fine sections were made on a Reichert-Jung ultramicrotome and ∗ Corresponding author. Tel.: +54 011 4524 8096; fax: +54 011 4524 8096. stained with toluidine blue, observed and photographed with a E-mail address: [email protected] (B.G. Galati). Motic digital light microscope. Ultrathin sections were stained with http://dx.doi.org/10.1016/j.aquabot.2014.11.002 0304-3770/© 2014 Elsevier B.V. All rights reserved. B.G. Galati et al. / Aquatic Botany 121 (2015) 46–51 47 Fig. 1. Live photograph of Cabomba caroliniana. (A) Submerged leaves (sl). (B) Detail of (A) where floating leaves (fl) can be easily distinguished while accompanying the flower. uranyl acetate and lead citrate (Zarlavsky, 2014), observed and photographed in a JEOL 1200 EX II. Fig. 2. Transverse sections of submerged and floating leaves observed with Light Free hand sections of fresh leaves were made and observed Microscope. (A) Floating leaf. (B) Submerged leaf. (C) Detail of A. d: diatoms; t: tri- and photographed with a fluorescence microscope Zeiss Axioplan, chome. (D) Detail of a floating leaf showing a stoma (st). (E) Detail of submerged in order to compare chlorophyll auto-fluorescence (wavelength leaf. Scale bars (A) and (B) 100 ␮m, (C), (D) and (E) 50 ␮m. 395 nm). For statistical analysis, the following variables were measured Epidermal chloroplasts are smaller and fewer than the ones in the epidermis and mesophyll chloroplasts: number of thylakoids present in the mesophyll (Fig. 3A). Chlorophyll observed in meso- per granum, wide of the chloroplast, and length of the chloro- phyll chloroplasts has a brighter fluorescence than chloroplasts in plast. Number of measurements for each of the variables studied the epidermal cells (Fig. 3B). is specified in Table 1. A parametric test (nominal significance level The epidermal and mesophyll chloroplasts have the same ˛ = 0.05) was performed after homogeneity of variance was ver- ultrastructural differences in both types of leaves. Mesophyll ified. A t-test was used since it allows testing the hypothesis on chloroplasts are, on average, 9.4 ␮m long and 4.5 ␮m wide. They the expectation of the random variable defined as a difference of have between 12 and 26 plastoglobuli and one or two starch sample means. It is assumed to have two independent samples: grains of 6.1 ␮m long and 2.5 ␮m wide on average (Fig. 4A and B). mesophyll and epidermis chloroplasts. The test is a tool for com- Grana are formed by an average of 29 thylakoids. Abundant inter- parison of means in two samples. All calculations were performed grana thylakoids are present in these chloroplasts (Fig. 4C). Many using the statistical software Infostat (Di Rienzo et al., 2009). mitochondria are observed around the chloroplasts of mesophyll cells (Fig. 4B). 3. Results Ribosomes and endoplasmic reticulum surround the epider- mal chloroplasts that are smaller than the ones of mesophyll cells The anatomy of floating and submerged leaves is similar (Fig. 1A (Fig. 5A–C). They are 4.4 ␮m long and 1.1 ␮m high on average. and B). The mesophyll consists of a single layer of cylindrical cells Between six and seven plastoglobuli are present in these chloro- with conspicuous and numerous chloroplasts (Fig. 2A–E). Floating plasts and starch grains are occasional and smaller (0.65 ␮m long leaves have larger intercellular spaces in this tissue. Small chloro- and 0.35 ␮m wide on average) (Fig. 5B and C). Grana have an aver- plasts are observed in the epidermis of both submerged and floating age of 9 thylakoids and intergrana thylakoids are scarce (Fig. 5 leaves. Stomata are present in the adaxial epidermis and tetracel- D). lular trichomes on the abaxial face of the floating leaves (Fig. 2A, C The parametric test results showed significant differences and D). (p < 0.0001) in the analyzed variables (number of thylakoids per 48 B.G. Galati et al. / Aquatic Botany 121 (2015) 46–51 Table 1 t-Test for independent samples. Comparative table of mean for number of thylakoids per granum, length of chloroplasts, and diameter of chloroplasts analyzed between mesophyll and epidermis chloroplasts; gl: degree of freedom; t: t-test. Characteristic Epidermis Mesophyll gl t p-Value n Median Variance n Median Variance Number of thylakoids 32 9.47 24.26 32 29.41 184.25 39 −7.81 <0.0001 Length of the chloroplast 15 1.11 0.11 15 4.58 1.93 34 −13.51 <0.0001 Wide of the chloroplast 15 4.47 0.88 15 9.4 17.16 35 −6.5 <0.0001 granum, wide of the chloroplast, and length of the chloroplast) of species (Esau, 1965; Mickel, 1972; Dickison, 2000). In Cabomba, the mesophyll chloroplasts with respect to the epidermis chloro- chloroplasts of the epidermis differ in size and ultrastructure from plasts (Table 1). Therefore, the number of thylakoids is higher in those present in the mesophyll. the mesophyll chloroplasts than in the epidermis chloroplasts, and Differences in size and ultrastructure of chloroplast in meso- mesophyll chloroplasts are wider and longer than the epidermis phyll and bundle sheath cells are common in C4 grasses and dicots chloroplasts. (Laetsch and Price, 1969; Johnson and Brown, 1973; Fisher and Evert, 1982; Sage and Monson, 1999). Cabomba presents dimor- 4. Discussion phism of chloroplasts similar to many C4 species. However, the C4 pathway first evolved 24–30 million years ago (Sage, 2004) The presence of chloroplasts in the leaf epidermis is common in and basal angiosperms have existed for about 130 million years aquatic plants and some terrestrial plants, especially shade adapted (Vialette-Guiraud et al., 2011) suggesting that Cabomba should be aC3 plant. There are some reports of chloroplasts polymorphism in some C3 species. Dimorphic chloroplasts were described in the dorsal root epidermis and leaf epidermis of Podostemoideae by Fujinami et al.